Using E. coli as a host organism in order to express, purify and characterize pGEM-gbr22
Introduction
Recombinant DNA (rDNA) has been a widespread source for research in both biological and biomedical science, specifically in the production of a specified protein through DNA implantation and expression [1]. Proteins that are in low abundance in their native organism can easily be purified for crystallography, enzyme inhibition assays, etc. [1,2]. Therefore, the most commonly used organism for over expressing the protein of interest is the bacteria E. Coli; A systematic method that is both cost effective and efficient and allows one to study human proteins without having to use human tissues. In addition, using E. coli allows for residues to occur and in turn allows for separation [1]. The protein production and purification process essentially calls for: obtaining the cDNA, cloning the cDNA into an E. coli expression vector, expressing the protein in a derivative of the E. coli BL21 (DE3) strain in a rich medium and good aeration, solubilizing and purifying the protein in a well-buffered solution, using immobilized metal affinity chromatography (IMAC) as the initial purification step, and if addition purification is required, using a size-exclusion chromatography (gel filtration) [2]. In this experiment, the plasmid (pGEM-gbr22), which encodes for a fluorescent protein, was produced and purified using E. coli BL21 (DE3) as the host bacteria strain for the expression and production of the intended specified protein. Doing so, it can be hypothesized that the pGEM-gbr22 protein will not yield a full 100 percent composition because the final purified protein product will essentially be composed of other contents.
Materials & Methods Protein Expression
Starting off, pGEM-gbr22 was first expressed in E. coli. The next step was transforming the expression strain of bacteria E. coli BL21 (DE3) with the plasmid. Doing so, 25ul of E. coli BL21 (DE3) was transferred into two transformation tubes. pGEM-gbr22 plasmid was added to the DNA tube and both tubes were heat shocked. SOC media was added to each of the tubes and were shaken in a water incubator for 30 minutes at 37 degrees Celsius at 250 rpm. 50ul of bacteria/SOC mixture was pipetted from each tube and was added to agar plates containing ampicillin, then taken to be incubated overnight. A single colony of bacteria was then added to LB/ampicillin media. The two tubes were incubated for 8 hours (37 degrees Celsius 200-350rpm). 0.625ml of starter culture containing pGEM-gbr22 was transferred into Erlenmeyer flask that contained LB and ampicillin. Thereafter, the flask was placed for twenty-four hours in the water shaker bath. A 500ul sample of culture was taken (sample 1). The remaining contents of the bacteria were poured into a 50ml conical tube and were centrifuged into an Allegra X-15 benchtop centrifuge (Beckman Coulter, Inc., Brea, CA) and was set to run for 10 minutes at 5000 rpm. The purple pellet at the bottom was saved and the remaining liquid was decanted. 2.5 ml of 1x PBS solution was mixed with the pellet. Lysozyme was also added to the tube and was stored in the -20 degrees Celsius freezer.
Protein purification
The next step calls for pGEM-gbr22 to be purified. 2ul of Benzonase was added. The lysate was distributed from the above procedure into two Eppendorf tubes and were then centrifuged. 50ul of supernatant was taken (sample2). The liquid supernatant from both tubes were transferred into a conical tube and filtered though a PES syringe filter. An Econo column was used to run the supernatant along with the Ni-NTA resin/buffer. In order to strip the Ni-NTA, 5 ml of wash was run through the column, then 5ml of elution, and 5ml of more elution. In addition, after capping the end, 1ml of 30 percent ethanol in nanopure water was added. The absorbance of the protein from elution 1 was measured using a Nanodrop spectrophotometer at 280 and 574 nanometers.
Protein characterization
Finally, the gbr22 protein was characterized and was done so by collecting the samples from the previous lab and preparing them using a 6x gel-loading buffer. Thereafter, the tubes of samples 1-6 were placed into a heat black at 95 degrees Celsius for five minutes and were then centrifuged for two minutes at 5000 rpm. Next, the SDS electrophoresis was assembled and prepared accordingly so that the samples could be run for twenty-five minutes at 200V. In the first well, the protein ladder was inputted followed by , samples one through six and then samples four, five, and six from a partner. Next, the gel was stained with imperial protein stain and was left in the orbital shaker for an hour and a half. The gel was then dedestained and was left overnight in water with a kimwipe. The following day, the gel was dried.
Results
Fig. 1 Control plate composed of E. coli BL21 colonies with ampicillin and were incubated at 37 degrees celsius for twenty-four hours. No colonies were produced because ampicillin antibiotic killed off the bacteria colonies.
Fig. 2 Experimental cell culture plate composed of E. coli BL21 colonies that express a gene that is resistant to ampicillin. The ampicillin killed off all of E. coli that did not express protein and was also incubated at 37 degrees celsius for twenty-four hours.
Fig. 3 Fun Plate composed of collected microorganisms of the saliva from the inside cheeck of one student and saliva of another. Plate was incubated for twenty-four hours at thirty-seven degrees Celsius beforehand.
Fig. 4 25ml of LB broth and ampicillin solution with E. coli BL21 bacteria that expresses pGEM-gbr22. Flasks were shaken for twenty-four hours at thirty-seven degrees celsius.
Fig. 5 Spun down E. coli bacteria that expresses pGEM-gbr22 and resulted to pellet forming at the bottom after centrifuging occurred. Supernatant was removed from conical tube. Pellet weighed 0.39 grams.
Fig. 6 Elution 1 (left) and Elution 2 (right). Majority of purified pGEM-gbr22 protein washed using elution buffer. Elution 2 contains what was left of remaining protein pGEM-gbr22 that was not obtained in elution 1. This elution was prepared the same way with elution buffer in a 15ml conical tube.
Fig. 7 Nanodrop spectrophotometer absorbance spectra analysis for Elution 1 sample at 280nm. The path length for the reading was set to 10 mm and absorbance read 0.563 mg/ml Beer's Law used to calculate concentration.
Fig. 8 Nanodrop spectrophotometer absorbance spectra analysis for second sample of Elution 1 at 280nm. The path length for the reading was set to 10 mm and absorbance reads 0.550 mg/ml. Beer's Law used to calculate concentration.
Fig 9 Fig. 7 Nanodrop spectrophotometer absorbance spectra analysis for third sample from Elution 1 at 574nm. Absorbance reads 0.140 mg/ml. Beer's Law used to calculate concentration.
Fig. 10 Fig. 7 Nanodrop spectrophotometer absorbance spectra analysis for fourth sample of Elution 1 at 574nm. Absorbance read 0.136 mg/ml. Beer's Law used to calculate concentration.
Fig. 11 SDS gel electrophoresis after staining. From left to right the first column represent the ladder. The next six columns contain samples one through six and the following three contain samples four through six from another partner. Each purple band represents a separate kind of protein that was found. The darkest band, represents the pGEM-gbr22.
Fig. 12 Molecular weight standard ladder used for SDS electrophoresis and analysis.
Discussion
Most of the pure protein was acquired. Gel bands were slightly distinctive in both sample fives but more so in the second sample five. The second sample six showed to have a smaller amount of purple bands and mirrors to having a purer protein. In this lab, lysozyme was used in order to breakdown the cell walls of E. coli. Benzonase is known to break down RNA and DNA, and therefore was added to essentially decrease viscosity of the solution so that it could be relatively easier to pipette. Sample 1 contained pure bacterial cells because it collected the cells right after they were harvested. Sample two contained the lysate and sample three had the waste that came from the first run of the column. Within sample four lies the cell pellet solution and samples five and six contained the elution buffer that was running from the same column. The use of the elution buffer allowed the protein pGEM-gbr22 to be collected into the conical tube and the wash buffer was utilized to clean out the pellet after the waste had been removed. Imidazole was used so that the protein could ultimately unbind from Ni-NTA and be collected into elution 1 and 2. Sources of error in this experiment could range from pipetting an incorrect amount of specific solution, misreading the protocol, incorrect prelab calculations, or using unsterilized equipment.
Conclusion
At the conclusion of this lab, the protein pGEM-gbr22 was expressed, purified, and characterized through the use of recombinant DNA and E. coli in order to produce the protein. Essentially, results from this experiment showed that a specified protein , pGEM-gbr22 in this case, can be purified using a host organism and find the molecular weight of the protein. Given that the procedures were done correctly, it was then possible for one to apply the information and data that was gathered throughout the experiment and successfully estimate the concentration of the protein pGEM-gbr22 in the protein solution. Future directions and/or applications to consider of this work to VDS research would be to include enzyme assays and thus determine possible drug targets from there.
References
[1] Graslund, S.; Nordlund, P.; Weigelt, J.; Hallberg, B. M.; Bray, J.; Gileadi, O.; Knapp, S.; Oppermann, U.; Arrowsmith, C.; Hui, R.; Ming, J.; Protein production and purification. Nature Methods. 2008, 5(2): 135-46.
Introduction
Recombinant DNA (rDNA) has been a widespread source for research in both biological and biomedical science, specifically in the production of a specified protein through DNA implantation and expression [1]. Proteins that are in low abundance in their native organism can easily be purified for crystallography, enzyme inhibition assays, etc. [1,2]. Therefore, the most commonly used organism for over expressing the protein of interest is the bacteria E. Coli; A systematic method that is both cost effective and efficient and allows one to study human proteins without having to use human tissues. In addition, using E. coli allows for residues to occur and in turn allows for separation [1]. The protein production and purification process essentially calls for: obtaining the cDNA, cloning the cDNA into an E. coli expression vector, expressing the protein in a derivative of the E. coli BL21 (DE3) strain in a rich medium and good aeration, solubilizing and purifying the protein in a well-buffered solution, using immobilized metal affinity chromatography (IMAC) as the initial purification step, and if addition purification is required, using a size-exclusion chromatography (gel filtration) [2]. In this experiment, the plasmid (pGEM-gbr22), which encodes for a fluorescent protein, was produced and purified using E. coli BL21 (DE3) as the host bacteria strain for the expression and production of the intended specified protein. Doing so, it can be hypothesized that the pGEM-gbr22 protein will not yield a full 100 percent composition because the final purified protein product will essentially be composed of other contents.
Materials & Methods
Protein Expression
Starting off, pGEM-gbr22 was first expressed in E. coli. The next step was transforming the expression strain of bacteria E. coli BL21 (DE3) with the plasmid. Doing so, 25ul of E. coli BL21 (DE3) was transferred into two transformation tubes. pGEM-gbr22 plasmid was added to the DNA tube and both tubes were heat shocked. SOC media was added to each of the tubes and were shaken in a water incubator for 30 minutes at 37 degrees Celsius at 250 rpm. 50ul of bacteria/SOC mixture was pipetted from each tube and was added to agar plates containing ampicillin, then taken to be incubated overnight. A single colony of bacteria was then added to LB/ampicillin media. The two tubes were incubated for 8 hours (37 degrees Celsius 200-350rpm). 0.625ml of starter culture containing pGEM-gbr22 was transferred into Erlenmeyer flask that contained LB and ampicillin. Thereafter, the flask was placed for twenty-four hours in the water shaker bath. A 500ul sample of culture was taken (sample 1). The remaining contents of the bacteria were poured into a 50ml conical tube and were centrifuged into an Allegra X-15 benchtop centrifuge (Beckman Coulter, Inc., Brea, CA) and was set to run for 10 minutes at 5000 rpm. The purple pellet at the bottom was saved and the remaining liquid was decanted. 2.5 ml of 1x PBS solution was mixed with the pellet. Lysozyme was also added to the tube and was stored in the -20 degrees Celsius freezer.
Protein purification
The next step calls for pGEM-gbr22 to be purified. 2ul of Benzonase was added. The lysate was distributed from the above procedure into two Eppendorf tubes and were then centrifuged. 50ul of supernatant was taken (sample2). The liquid supernatant from both tubes were transferred into a conical tube and filtered though a PES syringe filter. An Econo column was used to run the supernatant along with the Ni-NTA resin/buffer. In order to strip the Ni-NTA, 5 ml of wash was run through the column, then 5ml of elution, and 5ml of more elution. In addition, after capping the end, 1ml of 30 percent ethanol in nanopure water was added. The absorbance of the protein from elution 1 was measured using a Nanodrop spectrophotometer at 280 and 574 nanometers.
Protein characterization
Finally, the gbr22 protein was characterized and was done so by collecting the samples from the previous lab and preparing them using a 6x gel-loading buffer. Thereafter, the tubes of samples 1-6 were placed into a heat black at 95 degrees Celsius for five minutes and were then centrifuged for two minutes at 5000 rpm. Next, the SDS electrophoresis was assembled and prepared accordingly so that the samples could be run for twenty-five minutes at 200V. In the first well, the protein ladder was inputted followed by , samples one through six and then samples four, five, and six from a partner. Next, the gel was stained with imperial protein stain and was left in the orbital shaker for an hour and a half. The gel was then dedestained and was left overnight in water with a kimwipe. The following day, the gel was dried.
Results
Discussion
Most of the pure protein was acquired. Gel bands were slightly distinctive in both sample fives but more so in the second sample five. The second sample six showed to have a smaller amount of purple bands and mirrors to having a purer protein. In this lab, lysozyme was used in order to breakdown the cell walls of E. coli. Benzonase is known to break down RNA and DNA, and therefore was added to essentially decrease viscosity of the solution so that it could be relatively easier to pipette. Sample 1 contained pure bacterial cells because it collected the cells right after they were harvested. Sample two contained the lysate and sample three had the waste that came from the first run of the column. Within sample four lies the cell pellet solution and samples five and six contained the elution buffer that was running from the same column. The use of the elution buffer allowed the protein pGEM-gbr22 to be collected into the conical tube and the wash buffer was utilized to clean out the pellet after the waste had been removed. Imidazole was used so that the protein could ultimately unbind from Ni-NTA and be collected into elution 1 and 2. Sources of error in this experiment could range from pipetting an incorrect amount of specific solution, misreading the protocol, incorrect prelab calculations, or using unsterilized equipment.
Conclusion
At the conclusion of this lab, the protein pGEM-gbr22 was expressed, purified, and characterized through the use of recombinant DNA and E. coli in order to produce the protein. Essentially, results from this experiment showed that a specified protein , pGEM-gbr22 in this case, can be purified using a host organism and find the molecular weight of the protein. Given that the procedures were done correctly, it was then possible for one to apply the information and data that was gathered throughout the experiment and successfully estimate the concentration of the protein pGEM-gbr22 in the protein solution. Future directions and/or applications to consider of this work to VDS research would be to include enzyme assays and thus determine possible drug targets from there.
References
[1] Graslund, S.; Nordlund, P.; Weigelt, J.; Hallberg, B. M.; Bray, J.; Gileadi, O.; Knapp, S.; Oppermann, U.; Arrowsmith, C.; Hui, R.; Ming, J.; Protein production and purification. Nature Methods. 2008, 5(2): 135-46.
[2] Weill Cornell Medical College. Studying Proteins and Protein Purification. http://www-users.med.cornell.edu/~jawagne/proteins_&_purification.html (accessed Apr 16, 2012).